The enormous increase in demand for synthetic oligonucleotides, fueled by the advances in DNA technology over the last decades, has been accelerated by recent progress in sequencing and decoding whole genomes, particularly the human genome. A number of methods in molecular biology and DNA based diagnostics to amplify, detect, analyze and quantify nucleic acids are dependent on chemically synthesized oligonucleotides.
Some applications of synthetic oligonucleotides in molecular biology require the presence of a terminal phosphate moiety, which is introduced into the synthetic oligonucleotides using specially designed phosphorylation reagents. One major application of synthetic oligonucleotides with terminal 5′-phosphate moieties is the enzymatic ligation of the oligonucleotide to another oligonucleotide with a 3′-OH group. Ligation reactions take place when the terminal 5′-phosphate of one oligonucleotide annealed to a template DNA strand is joined to a 3′-hydroxyl group of a second strand of annealed oligonucleotide adjacent to the first by a ligase enzyme. Terminal 5′-phosphate moieties are also amenable to the ligase chain reaction, which is useful to determine the sequence of a DNA sample, to detect certain types of DNA or to detect single point mutations in disease genes. Terminal 5′-phosphates are also useful to those skilled in the art for the purpose of building longer oligonucleotides for molecular cloning and gene construction. Terminal phosphate groups are also known by those skilled in the art to be amenable to covalent attachment via standard dehydration reactions with reporter groups bearing alcoholic substituents. Finally, terminal phosphate groups improve the stability of synthetic oligonucleotides by reducing exonucleolytic degradation.
The current state of the art in oligonucleotide synthesis is automated solid phase synthesis using phosphoramidite chemistry, which, in particular, is based on the developments of McBride et al. (1983) Tetrahedron Letters 24:245-248 and Sinha et al. (1983) Tetrahedron Letters 24:5843-5846, each of which is incorporated herein by reference in its entirety. Phosphoramidite chemistry, together with related methods such as hydrogen phosphonate chemistry, has been extensively reviewed with respect to their uses in oligonucleotide chemistry by Beaucage et al. (1992) Tetrahedron 48:2223-2311, which is incorporated herein by reference in its entirety. During solid phase oligonucleotide synthesis, a series of nucleotide monomers are sequentially attached, via their phosphoramidite derivatives, in a predetermined order to either, depending on the direction of chain extension, the 5′-functional group or the 3′-functional group of the growing oligonucleotide strand. The oligonucleotide strand is anchored to an insoluble moiety such as controlled pore glass or polystyrene resin beads. The method of attachment of each monomer is generally comprised of the following steps:    1. Deprotection of the reactive functionality. The common reactive functionality is the 5′-hydroxyl group of the terminal nucleoside. This functionality is usually protected with a 4,4′-dimethoxytrityl (DMT) moiety that can be removed via acid treatment. One of the attractive features of the DMT moiety is that it forms a bright orange DMT cation during acid deprotection. This cation serves effectively as reporter group that can be easily monitored at a wavelength between 480 and 500 nm for the purpose of judging the completeness the previous coupling step. Most commercially available automated synthesizers have the capability to monitor the released DMT cation. This data gives the operator an instant indication of whether or not the synthesis failed at any given step.    2. Coupling by addition of a phosphoramidite derivative and an activator. The phosphoramidite derivative is usually a nucleoside phosphoramidite, however, it may also be a phosphoramidite derivatized with a different organic moiety.    3. Capping of unreacted terminal functional groups. This step introduces an inert protective group that prevents further coupling to failure sequences.    4. Oxidation of the newly formed phosphorous nucleotide backbone linkage from the trivalent phosphite to the stable pentavalent phosphate state.    5. After a washing step, the process is repeated.
The current state of the art in the introduction of terminal 5′-O-phosphate groups into synthetic oligonucleotides relies on phosphoramidite reagents that are employed in the final coupling cycle of the oligonucleotide synthesis. The preferred phosphoramidite reagents carry two phosphate protective groups that are removed via mild basic hydrolysis. In most variants of such phosphoramidite reagents one of the protective groups is a cyanoethyl group, an industry standard phosphate protective group when oligonucleotides are produced using phosphoramidite chemistry. The cyanoethyl protective group is removed under mild basic conditions after oxidation to the pentavalent phosphate species and is stable to the strongly acidic conditions employed in the deprotection step of oligonucleotide synthesis cycles. Cyanoethyl protective groups are removed concurrently by the final ammonium hydroxide treatment that is required to cleave the linkage of the oligonucleotide to the solid support. In commercially available phosphoramidite reagents for the introduction of terminal phosphate moieties the second protective group is also susceptible to base hydrolysis and contains a reporter group, usually a DMT group that can be removed by acid treatment. The significance of the DMT group is due to the common practice of monitoring amidite coupling efficiencies by measuring the release of the colored DMT cation from the previously coupled phosphoramidite derivative. The DMT cation is easily monitored by VIS-spectroscopy at a wavelength between 480 and 500 nm.
Cyanoethyl protective groups belong to a class of phosphate protective groups known as “β-eliminators.” β-Eliminators are phosphate protective groups that are removable with aqueous bases such as ammonium hydroxide. β-Eliminators are cleaved by a mechanism that does not involve the attack of a cleavage reagent at the phosphorus center. Therefore, phosphates protected with β-eliminating protective groups are not amenable to side reactions such as chain cleavage reactions, which is a known problem with phosphate protective groups that are cleaved by other mechanisms. Thus, most of the phosphate protective groups used in oligonucleotide synthesis are β-eliminators. Until now, however, there have been no examples of dual cyanoethyl-protected phosphoramidites that incorporate a reporter group. Additionally, there are no examples of β-eliminating phosphate protective groups that are functionalized at the alpha position.
The second protective group in the state of the art phosphoramidite reagents for the introduction of terminal phosphate groups can be either a different β-eliminating protective group or a protective group that requires additional manipulations before it is released. Either type of protective group also incorporates an ether linkage to a DMT group which, when cleaved by acid, serves as a reporter group. Horn et al., U.S. Pat. No. 5,252,760 and (1986) Tetrahedron Letters 27:4705-4708, each of which is specifically incorporated herein by reference in its entirety, have described the use of a state of the art chemical phosphorylation reagent that has one cyanoethyl group and a unique second β-eliminating group that contains a DMT reporter group and can be removed via mild basic hydrolysis. This phosphorylation reagent, however is a hard to handle viscous oil. This drawback is especially manifest due to the fact that very small amounts of reagent are required to be packaged in amber bottles when using current state of the art automated oligonucleotide synthesis. This makes assessment of whether or not the compound is fully dissolved very difficult.
Guzaev et al., U.S. Pat. No. 5,959,090, (1995) Tetrahedron 51:9375-9384 and (1999) Tetrahedron 55:9101-9116, each of which is specifically incorporated herein by reference in its entirety, have described the use of another state of the art phosphoramidite reagent for the introduction of terminal phosphate groups that carries one cyanoethyl group and a unique phosphate protective group containing the desired DMT reporter group. However, this phosphoramidite reagent requires two manipulations to be fully deprotected. The mechanism of deprotection requires that the DMT group be removed via acid treatment followed by mild basic treatment that leads to a retrograde aldol reaction followed by a β-elimination. It should be noted that complete detritylation must be realized using this reagent, otherwise, significant impurities will be present in the final product due to the partially protected terminal phosphate. Experimental results reported by the Guzaev et al. show that detritylation conditions must be stringent in order to ensure complete detritylation. At least two detritylation cycles on an automated oligonucleotide synthesizer must be employed when using this reagent. This can lead to further complexity in modern, high-throughput, automated oligonucleotide synthesizers as well as additional oligonucleotide impurities that are known to occur upon prolonged treatment with acid. It should also be noted that this reagent in its diester form is a viscous oil that suffers from the same problems noted for the Horn et al. reagent. This reagent in its bis(ethylamido) form is a solid, however, it suffers from the same deprotection issues as the diester form as well as requiring an inconvenient and long synthesis procedure. Recently, Guzaev et al. (2001) Tetrahedron Lett. 42:4769-4773, which is specifically incorporated herein by reference in its entirety, have disclosed an improvement to the aforementioned reagents by introducing a TMT (trimethoxytrityl) group instead of the formerly employed DMT-group. The TMT-group is more easily removed than the DMT-group under acidic conditions, but it is also less convenient for monitoring coupling efficiencies, because the spectral characteristics of the released TMT-cation differ from those of the DMT-cation.
A phosphoramidite reagent for the routine synthesis of oligonucleotides with terminal phosphate moieties should fulfill the following criteria:                A) It should contain a reporter group, preferably a DMT-group, that can be monitored calorimetrically at a wavelength between 480 and 500 nm;        B) It should contain two β-eliminating phosphate protective groups;        C) It should not require additional manipulations to effect final deprotection after the reagent is added to the oligonucleotide in the standard synthesis cycle; and        D) It should be a solid that can be easily manipulated by an operator and can be easily monitored with respect to whether or not the reagent is completely solubilized.        
Until now, there is no one reagent that meets all of the aforementioned criteria. The commercialized state of the art phosphoramidite reagent described by Horn et al. is a viscous oil that is difficult to monitor when dissolving. The reagent described by Guzaev et al. is also a viscous oil that requires additional manipulations to effect final deprotection. The bis(ethylamido)-derivative described by Guzaev et al. is a solid, however, it still requires additional manipulations to effect the final deprotection that yields the terminal 5′-O-phosphate monoester.
A variety of other phosphoramidite reagents for the introduction of terminal phosphate moieties in the last coupling cycle of an oligonucleotide synthesis have been described in the scientific literature. Examples include reagents with two allyl phosphate protective groups as described by Bannwarth et al. (1989) Tetrahedron Letters 30:4219-4222, a reagent with a methyl phosphate protective group and a tritylthioethyl phosphate protective group as described by Connolly (1987) Tetrahedron Letters 28:463-466, reagents with two 2-cyanoethyl phosphate protective groups or two p-nitrophenylethyl phosphate protective groups as described by Uhlmann et al. (1986) Tetrahedron Letters 27:1023-1026, and another reagent with two p-nitrophenylethyl phosphate protective groups as described by Schwarz et al. (1987) Nucleosides & Nucleotides 6:537-539. A comprehensive review of the field is described by Beaucage et al. (1993) Tetrahedron 49:10441-10488. Each of the references cited above is specifically incorporated herein by reference in its entirety.
None of the phosphoramidite reagents described in these references fulfills all of the criteria set forth above. None of the described reagents contain a reporter group useful for monitoring the efficiency of the coupling reaction of the reagent. In addition, they are viscous liquids which are difficult to place into vials or bottles and which are hard to visually monitor for complete dissolution in the solvent of the coupling reaction. Also, some of the reagents require an additional manipulation step in the deprotection of the synthetic oligonucleotide. For example, the p-nitrophenylethyl phosphate protective group requires treatment with the strong base DBU in addition to the standard ammonia treatment in order to achieve its complete removal. Another example is the tritylthioethyl group, which requires the removal of the trityl group from the sulfur atom with silver salts in addition to the standard ammonia treatment. Additionally, some of the described reagents contain phosphate protective groups that are cleaved through an attack of a deprotecting agent at the phosphorus atom, a mechanism that may result in dephosphorylation as a side reaction. For example, the methyl phosphate protective group employed in some of the described reagents is removed through an attack of strong nucleophiles at the phosphorus atom, which facilitates dephosphorylation as a side reaction.
There is a need for a phosphoramidite reagent that does not suffer from any of the aforementioned disadvantages and that combines all of the favorable features of an ideal phosphoramidite reagent for the introduction of terminal phosphate moieties into synthetic oligonucleotides. The present invention describes novel phosphoramidite reagents that combine all of the criteria set forth above for an ideal phosphoramidite reagent. The phosphoramidite reagents described herein contain a reporter group and comprise two β-eliminating phosphate protective groups, which are removable with ammonia and do not require an additional manipulation step for the deprotection of the synthesized oligonucleotide and that are solid compounds which easily dissolve in the solvent of the phosphoramidite coupling reaction. Included in the present invention are methods for the synthesis of oligonucleotides with terminal phosphate moieties using the phosphoramidite reagents of the invention.